The ubiquitin system is a major regulatory mechanism of cellular processes in which speed, specificity, and timing are critical. Ubiquitin-mediated proteolysis of key substrates controls cell cycle progression, signal transduction pathways, differentiation,apoptosis, DNA repair and the immune response. This process is mediated by a multimericmachine, composed of a regulatory ubiquitin-targeting component and an effector protein degradation engine. Theregulatory component, which targets ubiquitin to proteins destined for degradation, is itself composed of several multimeric elements (e.g., the SCFubiquitin ligase complexes) that contribute much of the specificity inherent in the process. In humans, there are sixty-eight SCFligases, each characterized by a different F-boxprotein subunit that provides specificity by directly recruiting the substrate to the rest of the ligase and,ultimately, to the ubiquitin-conjugating enzyme. Notably, only three out of 68 human SCFubiquitin ligases (containing the F-box proteins UTrcp, Fbw7 and Skp2, respectively) have well-established substrates, many of which are involved in cell cycle control. The remaining 65F-box proteins are considered as "orphan" since their substrates still await discovery. We have recently developed a novel immunopurification strategy that enriches for substrates of F-box proteins followed by mass spectrometry analysis. We will systematicallyidentify biologically significantsubstrates of human orphan F-boxproteins (Specific Aim 1). Because of our research interest, we will focus particularly on those orphan F-boxproteins that our preliminary results suggest to be involved in cell cycle control and cancer. Under Aim 2,we will validate the biologically most significantsubstrates identifiedunder Aim 1. Given their critical role in regulating cell proliferation, SCFligases are often the target of cancer- related deregulation and involved in oncogenic transformation. Therefore,the information gained from the proposed studies will be of direct relevance to cancer biology and other proliferative diseases.